十二烷基化壳聚糖基因支架血管内基因转运的实验研究

目的目前基因治疗方面存在的重要问题是缺乏有效的基因转运体系可以足量、安全地将治疗基因(无论病毒载体或非病毒载体)运送至体内靶细胞并提高其转染效率,以得到基因的高效表达。心血管内基因治疗的特殊性还在于很难把基因专一递送至血管组织的病灶处而不进入血液循环系统。实验研究提出了一种携带质粒DNA的烷基化壳聚糖纳米粒的血管内支架,可以有效地将质粒DNA递送至血管壁靶细胞并达到了高效转染的效果。     

mPEG-CS纳米粒介导livin shRNA基因纳米复合物的制备及转染效率

背景：作为非病毒基因转染载体,由可降解的高聚物形成的纳米载体目前被广泛由于基因转染,因为他们具有良好的缓释性,靶向性和生物相容性。 目的：制备mPEG-CS纳米粒,探讨mPEG-CS作为Livin shRNA基因转染载体的可行性。 方法：通过离子交联法制备mPEG-CS纳米粒,利用聚乙二醇对壳聚糖进行改性,通过静电吸附法制备载livin shRNA的基因纳米复合物。Zeta-size分析仪和透射电镜检测空白纳米粒和载livin shRNA的基因纳米复合物的形态、粒径和zeta电位,测定基因纳米复合物的包封率,凝胶电泳阻滞实验和DNase I酶消化实验验证纳米粒对基因的保护作用。利用最佳条件下制备的基因纳米复合物,转染大肠癌HT-29细胞,考察转染效率。 结果及结论：成功制备出约60 nm的mPEG-CS纳米粒,当纳米粒与基因体积比为3∶1时,得到的基因纳米复合物形较规则,粒径100 nm左右;其包封率为(94.32±0.35)%。凝胶电泳阻滞实验表明纳米粒能够紧密结合DNA,对基因具有良好的基因保护作用。该基因纳米复合物转染大肠癌细胞的转染效率高,持续作用时间长。mPEG-CS纳米粒作为基因转染载体,对基因具有保护作用,能够将livin shRNA重组质粒高效转染入大肠癌细胞,能够在大肠癌细胞内长时间表达,克服了RNA干扰在基因治疗肿瘤中基因作用时间较短的缺点。     

载基因壳聚糖纳米粒的研究

RNA干扰技术已被广泛应用于心血管领域,壳聚糖纳米粒以其良好的生物特性而作为基因递送载体成为现在研究的热点.就BNA干扰技术与纳米技术在心血管领域的应用及目前常采用的制备壳聚糖纳米粒的方法、影响质粒与壳聚糖纳米粒结合效率的因素、质粒壳聚糖复合物纳米粒转染的影响因素及体外释药行为作一简单的回顾.     

腺相关病毒载体应用的研究进展

以腺相关病毒作为基因治疗载体近年来备受关注。通过提高腺相关病毒载体的产量 ,扩大其载体容量 ,提高基因的转染效率和克服体内的免疫反应等手段 ,使腺相关病毒载体的应用更加符合临床的需要。     

地中海贫血的基因治疗

地贫的基因治疗,设想采用反转录病毒载体通过包装细胞系,转染MEL细胞和转染骨髓干细胞后植入患者体内,以获得珠蛋白基因细胞和整体水平的高表达。但目前主要问题是转移基因的表达水平低、病毒滴度低及前病毒的缺失与重排,其基因治疗仍处于载体构建阶段。     

基因治疗中的核酸药物及非病毒递送载体的研究进展

基因治疗是针对基因异常相关疾病的终极治疗技术,各种具有不同机制的核酸药物的出现为基因治疗带来了更多的可能性。但是,由于存在体内稳定性差、难以高效进入靶细胞等问题,核酸药物需要载体的帮助而进入目标细胞并到达特定的胞内位置,因此,开发安全高效的核酸递送系统是基因治疗的基石。与病毒载体相比,非病毒载体具有更高的安全性,但转染效率较低。随着纳米技术的发展,非病毒载体的效率得到了显著的提升,进入临床研究的数量逐渐增多。本文简要介绍基因治疗中的核酸药物及其递送载体,对非病毒核酸药物递送技术的瓶颈及进展做综合评述。     

地中海贫血的基因治疗

地贫的基因治疗，设想采用反转录病毒载体通过包装细胞系，转染ＭＥＬ细胞和转染骨髓干细胞后植入患者体内，以获得球蛋白基因细胞和整体水平的高表达。但目前主要问题是转移基因的表达水平低、病毒滴度低及前病毒的缺失与重排，其基因治疗仍处于载体构建阶段。     

Duchenne肌营养不良基因缺陷及基因治疗

Duchenne肌营养不良(DMD)是常见的神经肌肉遗传病之一,由于骨胳肌肌膜上的抗肌萎缩蛋白(dystrophin)完全或部分缺失引起.本文介绍了dystrophin的结构和功能,对DMD基因治疗的目的基因,基因治疗方式(包括病毒载体和非病毒载体),基因转染途径作了较为全面的介绍,指出腺相关病毒载体介导的基因治疗及干细胞移植是有希望的治疗方向,经全身途径使目的基因广泛转染骨胳肌并实现心肌和膈肌的转染,是基因治疗研究的难点.     

病毒载体介导的基因转移研究进展

实现基因治疗的关键在于目的基因的高效转移并适度表达 ,这将直接影响其治疗效率和安全性。因此探索理想的基因转移技术是基因治疗的一项重要内容 ,目前人们的注意力更多地集中于病毒载体 ,传统的逆转录病毒载体不能转染非分裂细胞 ,且载导容量有限 ,促使人们在寻找改进措施的同时积极研制其它类型的病毒载体 ,本文将阐述这些病毒载体的特性、应用情况及研究进展。     

非病毒型载体介导基因转染

基因载体是制约基因转移技术发展的关键。近年来,非病毒载体由于其安全、低毒、低免疫原性等特点而备受青睐。文章以脂质体和聚乙烯亚胺为代表,介绍了非病毒载体的性质、介导转染的机制。随着人们对细胞转染机制了解的深入以及生物材料科学的迅速发展,非病毒型载体将有望实现高效、低毒、靶向特异等特点,从而成为基因治疗中的理想载体。     

A ligand-mediated nanovector for targeted gene delivery and transfection in cancer cells

As conventional cancer therapies struggle with toxicity issues and irregular remedial efficacy, the preparation of novel gene therapy vectors could offer clinicians the tools for addressing the genetic errors of diseased tissue. The transfer of gene therapy to the clinic has proven difficult due to safety, target specificity, and transfection efficiency concerns. Polyethylenimine (PEI) nanoparticles have been identified as promising gene carriers that induce gene transfection with high efficiency. However, the inherent toxicity of the material and non-selective delivery are the major concerns in applying these particles clinically. Here, a non-viral nanovector has been developed by PEGylation of DNA-complexing PEI in nanoparticles functionalized with an Alexa Fluor 647 near infrared fluorophore, and the chlorotoxin (CTX) peptide which binds specifically to many forms of cancer. With this nanovector, the potential toxicity to healthy cells is minimized by both the reduction of the toxicity of PEI with the biocompatible copolymer and the targeted delivery of the nanovector to cancer cells, as evaluated by viability studies. The nanovector demonstrated high levels of targeting specificity and gene transfection efficiency with both C6 glioma and DAOY medulloblastoma tumor cells. Significantly, with the CTX as the targeting ligand, the nanovector may serve as a widely applicable gene delivery system for a broad array of cancer types.     

Acid-degradable cationic methacrylamide polymerized in the presence of plasmid DNA as tunable non-viral gene carrier

New acid-degradable cationic nanoparticles were synthesized using a monomer-to-polymer approach, which enabled highly flexible nanoparticle fabrication to obtain controlled properties such as size and conjugation with additional functionalities. The nanoparticles were designed to cause swelling and osmotic destabilization of the endosome, while cationic branches holding anionic DNA are cleaved from the polymeric backbone of the nanoparticles and make plasmid DNA accessible for efficient gene expression. Efficient release of plasmid DNA upon hydrolysis of the nanoparticles at the endosomal pH 5.0 and transportation of the released DNA to the nucleus of a cell were shown. In vitro studies showed significantly higher transfection efficiency by the degradable nanoparticles than polyethylenimine (PEI) polyplexes at very low concentrations (i.e., ng/mL). Size-dependent selective transfection of phagocytic cells (e.g., RAW 309 macrophages) and non-phagocytic cells (e.g., NIH 3T3 fibroblasts) was also achieved by using nanoparticles of two different sizes (240 nm and 680 nm in diameter), which implies feasibility of tunable gene therapy and DNA vaccination using the nanoparticle system. Preliminary pulmonary transfection of mice using the degradable nanoparticles demonstrated a remarkably higher expression of firefly luciferase at 70% lower concentration than using naked DNA alone. Implications and further improvement of the nanoparticles to be used in gene therapy are also discussed.     

The effect of nocodazole on the transfection efficiency of lipid-bilayer coated gold nanoparticles

Nonviral vectors are safer than viral systems for gene therapy applications. However, the limited efficacy always prevents their being widely used in clinical practice. Aside from searching new gene nonviral vectors, many researchers focus on finding out new substances to improve the transfection efficiency of existent vectors. In this work, we found a transfection enhancer, nocodazole (NCZ), for dimethyldioctadecylammonium (DODAB, a cationic lipid) bilayer coated gold nanoparticles (AuNPs) mediated gene delivery. It was found that NCZ produces 3-fold transfection enhancement to HEK 293T cells assessed by flow cytometry (FCM). The result was further confirmed by luciferase assay, in which NCZ induced more than 5 times improvement in transfection efficiency after 48 h of transfection. The results from the inductively coupled plasma mass spectrometry (ICP-MS) and FCM showed that NCZ did not affect the internalization of DODAB–AuNPs/DNA complexes. The trafficking of the complexes by transmission electron microscopy (TEM) indicated that the interrupted transportation of the complexes to the lysosomes contributed greatly to the transfection enhancement. Therefore, NCZ can be used as a transfection enhancer in DODAB–AuNPs mediated transfection system. This work also gave an insight to improving the efficiency of lipid-mediated transfection: modifying lipid on gold nanoparticles and pre-treating cells by NCZ before the transfection.     

Enhancement of chitosan nanoparticle-facilitated gene transfection by ultrasound both in vitro and in vivo

In recent years, inefficiency of transfection and the lack of safe gene vectors have limited the feasibility of gene therapy. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapies. Herein, we complexed chitosan to plasmids at various N/P ratios, the molar ratios of the amino groups of chitosan to the phosphate groups of DNA, to create chitosan-DNA nanoparticles (CDNs), and then measured CDNs size, zeta-potential, efficiency of plasmid complexation, and plasmid integrity from enzyme digestion. We also used flow cytometry and fluorescence microscopy to examine the effect of an ultrasound (US) regimen on the efficiency of transfection of HeLa cells. The results revealed that the average size, zeta-potential, and loading efficiency of plasmid DNA in CDNs were 180-200 nm, 26-35 mV, and greater than 80%, respectively. Moreover, the transgene expression could be enhanced efficiently while HeLa cells or tumor tissues were given CDNs and then treated with US. Therefore, the use of chitosan nanoparticles and an US regimen shows great promise as an effective method of gene therapy. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.     

Chitosan DNA nanoparticles for oral gene delivery

Gene therapy is a promising technology with potential applications in the treatment of medical conditions, both congenital and acquired. Despite its label as breakthrough technology for the 21^st century, the simple concept of gene therapy - the introduction of a functional copy of desired genes in affected individuals - is proving to be more challenging than expected. Oral gene delivery has shown intriguing results and warrants further exploration. In particular, oral administration of chitosan DNA nanoparticles, one the most commonly used formulations of therapeutic DNA, has repeatedly demonstrated successful in vitro and in vivo gene transfection. While oral gene therapy has shown immense promise as treatment options in a variety of diseases, there are still significant barriers to overcome before it can be considered for clinical applications. In this review we provide an overview of the physiologic challenges facing the use of chitosan DNA nanoparticles for oral gene delivery at both the extracellular and intracellular level. From administration at the oral cavity, chitosan nanoparticles must traverse the gastrointestinal tract and protect its DNA contents from significant jumps in pH levels, various intestinal digestive enzymes, thick mucus layers with high turnover, and a proteinaceous glycocalyx meshwork. Once these extracellular barriers are overcome, chitosan DNA nanoparticles must enter intestinal cells, escape endolysosomes, and disassociate from genetic material at the appropriate time allowing transport of genetic material into the nucleus to deliver a therapeutic effect. The properties of chitosan nanoparticles and modified nanoparticles are discussed in this review. An understanding of the barriers to oral gene delivery and how to overcome them would be invaluable for future gene therapy development.     

Chondrogenesis of human mesenchymal stem cells mediated by the combination of SOX trio SOX5, 6, and 9 genes complexed with PEI-modified PLGA nanoparticles

Target gene transfection for desired cell differentiation has recently become a major issue in stem cell therapy. For the safe and stable delivery of genes into human mesenchymal stem cells (hMSCs), we employed a non-viral gene carrier system such as polycataionic polymer, poly(ethyleneimine) (PEI), polyplexed with a combination of SOX5, 6, and 9 fused to green fluorescence protein (GFP), yellow fluorescence protein (YFP), or red fluorescence protein (RFP) coated onto PLGA nanoparticles. The transfection efficiency of PEI-modified PLGA nanoparticle gene carriers was then evaluated to examine the potential for chondrogenic differentiation by carrying the exogenous SOX trio (SOX5, 6, and 9) in hMSCs. Additionally, use of PEI-modified PLGA nanoparticle gene carriers was evaluated to investigate the potential for transfection efficiency to increase the potential ability of chondrogenesis when the trio genes (SOX5, 6, and 9) polyplexed with PEI were delivered into hMSCs. SOX trio complexed with PEI-modified PLGA nanoparticles led to a dramatic increase in the chondrogenesis of hMSCs in in vitro culture systems. For the PEI/GFP and PEI/SOX5, 6, and 9 genes complexed with PLGA nanoparticles, the expressions of GFP as reporter genes and SOX9 genes with PLGA nanoparticles showed 80% and 83% of gene transfection ratios into hMSCs two days after transfection, respectively.     

超声－微气泡介导的基因转染研究进展

能否成功高效地转染靶基因对于基因治疗的效果具有决定性的影响。微气泡是具有稳定的封装壳,直径为微米量级的小气泡,已作为超声造影剂被广泛应用。微气泡在超声脉冲的作用下可以在靶区释放其携带的基因并使之转染,同时超声脉冲产生的热效应和空化效应能提高转染率。若将微气泡与磁性纳米颗粒结合,还可以进一步提高转染率和转染精度,是一种理想、安全的基因载体。本文综述了由超声－微气泡介导的基因转染在近5年的主要研究成果。对影响转染率的主要因素如微气泡种类、超声辐照条件、基因及受体类型等方面做了详细的论述,并对微气泡介导基因转染过程中的安全性、长效性和差异性等问题进行了讨论。     

Synthesis and efficient hepatocyte targeting of galactosylated chitosan as a gene carrier in vitro and in vivo

While chitosan (CS) has been researched widely as a non-viral vector, its usefulness has been limited by its low cell specificity and transfection efficiency. Therefore, we successfully synthesized galactosylated chitosan (GC) and complexed it with an enhanced green fluorescent protein plasmid (pIRES-EGFP) for transfection into cultured H22 cells (murine hepatic cancer cell line) using various GC/EGFP (N/P) charge ratios. Maximal gene transfection rates detected by flow cytometry occurred at an N/P ratio 5:1. Compared with those of lipofectin/EGFP and naked pIRES-EGFP, GC/EGFP complexes show a very efficient cell-selective transfection to hepatocytes. The MTT assay detected relatively low cytotoxicity in cells transfected with GC. A recombinant plasmid granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin (IL) 21 (pIRES/GM-CSF-IL21) was successfully constructed and GC/GM-CSF-IL21 nanoparticles (average diameter, 82.1 nm) were administered via the tail vein of mice with liver metastasis of colon cancer model, for 5 consecutive days. The GC/GM-CSF-IL21 nanoparticles exhibited hepatocyte and passive tumor specificity, increased therapeutic efficacy compared to control groups, promoted leukocytes to aggregate in tumor tissues, and activated the cytotoxicity of natural killer (NK) cells and cytolytic T lymphocyte (CTL). Our results indicate that GC can be used in gene therapy to improve transfection efficiency and can be used as an immunological stimulant in vivo.     

A general strategy to achieve ultra-high gene transfection efficiency using lipid-nanoparticle composites

Gene therapy provides a new hope for previously “incurable” diseases. Low gene transfection efficiency, however, is the bottle-neck to the success of gene therapy. It is very challenging to develop non-viral nanocarriers to achieve ultra-high gene transfection efficiencies. Herein, we report a novel design of “tight binding-but-detachable” lipid-nanoparticle composite to achieve ultrahigh gene transfection efficiencies of 60∼82%, approaching the best value (∼90%) obtained using viral vectors. We show that Fe@CNPs nanoparticles coated with LP-2000 lipid molecules can be used as gene carriers to achieve ultra-high (60–80%) gene transfection efficiencies in HeLa, U-87MG, and TRAMP-C1 cells. In contrast, Fe@CNPs having surface-covalently bound N,N,N-trimethyl-N-2-methacryloxyethyl ammonium chloride (TMAEA) oligomers can only achieve low (23–28%) gene transfection efficiencies. Similarly ultrahigh gene transfection/expression was also observed in zebrafish model using lipid-coated Fe@CNPs as gene carriers. Evidences for tight binding and detachability of DNA from lipid-nanoparticle nanocarriers will be presented.     

Efficient transfection method using deacylated polyethylenimine-coated magnetic nanoparticles

Low efficiencies of nonviral gene vectors, such as transfection reagent, limit their utility in gene therapy. To overcome this disadvantage, we report on the preparation and properties of magnetic nanoparticles [diameter (d) = 121.32 ± 27.36 nm] positively charged by cationic polymer deacylated polyethylenimine (PEI max), which boosts gene delivery efficiency compare with polyethylenimine (PEI), and their use for the forced expression of plasmid delivery by application of a magnetic field. Magnetic nanoparticles were coated with PEI max, which enabled their electrostatic interaction with negatively charged molecules such as plasmid. We successfully transfected 81.1 ± 4.0% of the cells using PEI max-coated magnetic nanoparticles (PEI max-nanoparticles). Along with their superior properties as a DNA delivery vehicle, PEI max-nanoparticles offer to deliver various DNA formulations in addition to traditional methods. Furthermore, efficiency of the gene transfer was not inhibited in the presence of serum in the cells. PEI max-nanoparticles may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.